T-box 2, a mediator of Bmp-Smad signaling, induced hyaluronan synthase 2 and Tgfbeta2 expression and endocardial cushion formation.
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ABSTRACT:
SUBMITTER: Shirai M
PROVIDER: S-EPMC2773962 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Transformation of atrioventricular (AV) canal endocardium into invasive mesenchyme correlates spatially and temporally with the expression of bone morphogenetic protein (BMP)-2 in the AV myocardium. We revealed the presence of mRNA of Type I BMP receptors, BMPR-1A (ALK3), BMPR-1B (ALK6) and ALK2 in chick AV endocardium at stage-14(-), the onset of epithelial to mesenchymal transformation (EMT), by RT-PCR and localized BMPR-1B mRNA in the endocardium by in situ hybridization. To circumvent the functional redundancies among the Type I BMP receptors, we applied dominant-negative (dn) BMPR-1B-viruses to chick AV explants and whole-chick embryo cultures to specifically block BMP signaling in AV endocardium during EMT. dnBMPR-1B-virus infection of AV endocardial cells abolished BMP-2-supported AV endocardial EMT. Conversely, caBMPR-1B-virus infection promoted AV endocardial EMT in the absence of AV myocardium. Moreover, dnBMPR-1B-virus treatments significantly reduced myocardially supported EMT in AV endocardial-myocardial co-culture. AV cushion mesenchymal cell markers, alpha-smooth muscle actin (SMA), and TGFbeta3 in the endocardial cells were promoted by caBMPR-1B and reduced by dnBMPR-1B infection. Microinjection of the virus into the cardiac jelly in the AV canal at stage-13 in vivo (ovo) revealed that the dnBMPR-1B-virus-infected cells remained in the endocardial epithelium, whereas caBMPR-1B-infected cells invaded deep into the cushions. These results provide evidence that BMP signaling through the AV endocardium is required for the EMT and the activation of the BMP receptor in the endocardium can promote AV EMT in the chick.
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Project description:A key step in heart development is the coordinated development of the atrioventricular canal (AVC), the constriction between the atria and ventricles that electrically and physically separates the chambers, and the development of the atrioventricular valves that ensure unidirectional blood flow. Using knock-out and inducible overexpression mouse models, we provide evidence that the developmentally important T-box factors Tbx2 and Tbx3, in a functionally redundant manner, maintain the AVC myocardium phenotype during the process of chamber differentiation. Expression profiling and ChIP-sequencing analysis of Tbx3 revealed that it directly interacts with and represses chamber myocardial genes, and induces the atrioventricular pacemaker-like phenotype by activating relevant genes. Moreover, mutant mice lacking 3 or 4 functional alleles of Tbx2 and Tbx3 failed to form atrioventricular cushions, precursors of the valves and septa. Tbx2 and Tbx3 trigger development of the cushions through a regulatory feed-forward loop with Bmp2, thus providing a mechanism for the co-localization and coordination of these important processes in heart development.
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Project description:Distal outgrowth and maturation of mesenchymalized endocardial cushions are critical morphogenetic events during post-EMT atrioventricular (AV) valvuloseptal morphogenesis. We explored the role of BMP-2 in the regulation of valvulogenic extracellular matrix (ECM) components, versican and hyaluronan (HA), and cell migration during post-EMT AV cushion distal outgrowth/expansion. We observed intense staining of versican and HA in AV cushion mesenchyme from the early cushion expansion stage, Hamburger and Hamilton (HH) stage-17 to the cushion maturation stage, HH stage-29 in the chick. Based on this expression pattern we examined the role of BMP-2 in regulating versican and HA using 3D AV cushion mesenchymal cell (CMC) aggregate cultures on hydrated collagen gels. BMP-2 induced versican expression and HA deposition as well as mRNA expression of versican and Has2 by CMCs in a dose dependent manner. Noggin, an antagonist of BMP, abolished BMP-2-induced versican and HA as well as mRNA expression of versican and Has2. We further examined whether BMP-2-promoted cell migration was associated with expression of versican and HA. BMP-2- promoted cell migration was significantly impaired by treatments with versican siRNA and HA oligomer. In conclusion, we provide evidence that BMP-2 induces expression of versican and HA by AV CMCs and that these ECM components contribute to BMP-2-induced CMC migration, indicating critical roles for BMP-2 in distal outgrowth/expansion of mesenchymalized AV cushions.
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Project description:Endocardial cushion defect (ECD) can be partial (with two distinct valves) or complete (only one atrioventricular valve), and surgical therapy is usually required. The optimal surgical technique is controversial but De Vega's annuloplasty is widely performed. Tricuspid valve thrombosis are rarely seen after surgery. We present a 39-year-old male patient with tricuspid valve thrombosis after De Vega's annuloplasty without the use of a ring.
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Project description:Function of the heart begins long before its formation is complete. Analyses in mouse and zebrafish have shown that myocardial function is not required for early steps of organogenesis, such as formation of the heart tube or chamber specification. However, whether myocardial function is required for later steps of cardiac development, such as endocardial cushion (EC) formation, has not been established. Recent technical advances and approaches have provided novel inroads toward the study of organogenesis, allowing us to examine the effects of both genetic and pharmacological perturbations of myocardial function on EC formation in zebrafish. To address whether myocardial function is required for EC formation, we examined silent heart (sih(-/-)) embryos, which lack a heartbeat due to mutation of cardiac troponin T (tnnt2), and observed that atrioventricular (AV) ECs do not form. Likewise, we determined that cushion formation is blocked in cardiofunk (cfk(-/-)) embryos, which exhibit cardiac dilation and no early blood flow. In order to further analyze the heart defects in cfk(-/-) embryos, we positionally cloned cfk and show that it encodes a novel sarcomeric actin expressed in the embryonic myocardium. The Cfk(s11) variant exhibits a change in a universally conserved residue (R177H). We show that in yeast this mutation negatively affects actin polymerization. Because the lack of cushion formation in sih- and cfk-mutant embryos could be due to reduced myocardial function and/or lack of blood flow, we approached this question pharmacologically and provide evidence that reduction in myocardial function is primarily responsible for the defect in cushion development. Our data demonstrate that early myocardial function is required for later steps of organogenesis and suggest that myocardial function, not endothelial shear stress, is the major epigenetic factor controlling late heart development. Based on these observations, we postulate that defects in cardiac morphogenesis may be secondary to mutations affecting early myocardial function, and that, in humans, mutations affecting embryonic myocardial function may be responsible for structural congenital heart disease.
| S-EPMC406391 | biostudies-literature
Project description:Endocardial cushion formation is essential for heart valve development and heart chamber separation. Abnormal endocardial cushion formation often causes congenital heart defects. β-Catenin is known to be essential for endocardial cushion formation; however, the underlying cellular and molecular mechanisms remain incompletely understood. Here, we show that endothelial-specific deletion of β-catenin in mice resulted in formation of hypoplastic endocardial cushions due to reduced cell proliferation and impaired cell migration. By using a β-catenin DM allele in which the transcriptional function of β-catenin is selectively disrupted, we further reveal that β-catenin regulated cell proliferation and migration through its transcriptional and non-transcriptional function, respectively. At the molecular level, loss of β-catenin resulted in increased expression of cell cycle inhibitor p21 in cushion endocardial and mesenchymal cells in vivo. In vitro rescue experiments with HUVECs and pig aortic valve interstitial cells confirmed that β-catenin promoted cell proliferation by suppressing p21. In addition, one savvy negative observation is that β-catenin was dispensable for endocardial-to-mesenchymal fate change. Taken together, our findings demonstrate that β-catenin is essential for cell proliferation and migration but dispensable for endocardial cells to gain mesenchymal fate during endocardial cushion formation. Mechanistically, β-catenin promotes cell proliferation by suppressing p21. These findings inform the potential role of β-catenin in the etiology of congenital heart defects.
| S-EPMC10310929 | biostudies-literature
Project description:Distal outgrowth, maturation and remodeling of the endocardial cushion mesenchyme in the atrioventricular (AV) canal are the essential morphogenetic events during four-chambered heart formation. Mesenchymalized AV endocardial cushions give rise to the AV valves and the membranous ventricular septum (VS). Failure of these processes results in several human congenital heart defects. Despite this clinical relevance, the mechanisms governing how mesenchymalized AV endocardial cushions mature and remodel into the membranous VS and AV valves have only begun to be elucidated. The role of BMP signaling in the myocardial and secondary heart forming lineage has been well studied; however, little is known about the role of BMP2 expression in the endocardial lineage. To fill this knowledge gap, we generated Bmp2 endocardial lineage-specific conditional knockouts (referred to as Bmp2 cKOEndo) by crossing conditionally-targeted Bmp2flox/flox mice with a Cre-driver line, Nfatc1Cre, wherein Cre-mediated recombination was restricted to the endocardial cells and their mesenchymal progeny. Bmp2 cKOEndo mouse embryos did not exhibit failure or delay in the initial AV endocardial cushion formation at embryonic day (ED) 9.5-11.5; however, significant reductions in AV cushion size were detected in Bmp2 cKOEndo mouse embryos when compared to control embryos at ED13.5 and ED16.5. Moreover, deletion of Bmp2 from the endocardial lineage consistently resulted in membranous ventricular septal defects (VSDs), and mitral valve deficiencies, as evidenced by the absence of stratification of mitral valves at birth. Muscular VSDs were not found in Bmp2 cKOEndo mouse hearts. To understand the underlying morphogenetic mechanisms leading to a decrease in cushion size, cell proliferation and cell death were examined for AV endocardial cushions. Phospho-histone H3 analyses for cell proliferation and TUNEL assays for apoptotic cell death did not reveal significant differences between control and Bmp2 cKOEndo in AV endocardial cushions. However, mRNA expression of the extracellular matrix components, versican, Has2, collagen 9a1, and periostin was significantly reduced in Bmp2 cKOEndo AV cushions. Expression of transcription factors implicated in the cardiac valvulogenesis, Snail2, Twist1 and Sox9, was also significantly reduced in Bmp2 cKOEndo AV cushions. These data provide evidence that BMP2 expression in the endocardial lineage is essential for the distal outgrowth, maturation and remodeling of AV endocardial cushions into the normal membranous VS and the stratified AV valves.
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Project description:Cardiac valves are essential to direct forward blood flow through the cardiac chambers efficiently. Congenital valvular defects are prevalent among newborns and can cause an immediate threat to survival as well as long-term morbidity. Valve leaflet formation is a rigorously programmed process consisting of endocardial epithelial-mesenchymal transformation (EMT), mesenchymal cell proliferation, valve elongation and remodeling. Currently, little is known about the coordination of the diverse signals that regulate endocardial cushion development and valve elongation. Here, we report that the T-box transcription factor Tbx20 is expressed in the developing endocardial cushions and valves throughout heart development. Ablation of Tbx20 in endocardial cells causes severe valve elongation defects and impaired cardiac function in mice. Our study reveals that endocardial Tbx20 is crucial for valve endocardial cell proliferation and extracellular matrix development, but is not required for initiation of EMT. Elimination of Tbx20 also causes aberrant Wnt/?-catenin signaling in the endocardial cushions. In addition, Tbx20 regulates Lef1, a key transcriptional mediator for Wnt/?-catenin signaling, in this developmental process. Our study suggests a model in which Tbx20 regulates the Wnt pathway to direct endocardial cushion maturation and valve elongation, and provides new insights into the etiology of valve defects in humans.
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Project description:Comparison of differential gene expression between controls and mice that lack the transciption factor KLF2 from the cardiac endocardium following Nfatc1 Cre deletion.
2017-11-06 | GSE80964 | GEO
Project description:Ezh2 is a histone trimethyltransferase that silences genes mainly via catalyzing trimethylation of histone 3 lysine 27 (H3K27Me3). The role of Ezh2 as a regulator of gene silencing and cell proliferation in cancer development has been extensively investigated; however, its function in heart development during embryonic cardiogenesis has not been well studied. In the present study, we used a genetically modified mouse system in which Ezh2 was specifically ablated in the mouse heart. We identified a wide spectrum of cardiovascular malformations in the Ezh2 mutant mice, which collectively led to perinatal death. In the Ezh2 mutant heart, the endocardial cushions (ECs) were hypoplastic and the endothelial-to-mesenchymal transition (EMT) process was impaired. The hearts of Ezh2 mutant mice also exhibited decreased cardiomyocyte proliferation and increased apoptosis. We further identified that the Hey2 gene, which is important for cardiomyocyte proliferation and cardiac morphogenesis, is a downstream target of Ezh2. The regulation of Hey2 expression by Ezh2 may be independent of Notch signaling activity. Our work defines an indispensible role of the chromatin remodeling factor Ezh2 in normal cardiovascular development.
| S-EPMC3270034 | biostudies-literature